Secondary active transporters are a class of membrane proteins that utilize pre-existing molecular concentration gradients as an energy source for translocating another substrate, such as a nutrient or a neurotransmitter, against its concentration gradient. This process requires the protein to change conformations so as to expose a pathway to the substrate binding site(s) on one or other side of the membrane, in a cycle known as alternating access. Every organism expresses dozens of different secondary transporter proteins, and these exhibit a diverse set of architectures, albeit always with some form of internal structural symmetry. Unprecedented, ground-breaking insights have been garnered from three-dimensional structures obtained in the last decade. Nevertheless, a detailed understanding of the mechanism of each membrane transport protein requires knowledge of its structure in many more conformational states, including identification of the binding regions for the substrate or substrates. Moreover, those structures need to be placed into a context of dynamic ensembles on a thermodynamic landscape separated by kinetic barriers. Studies from our group over the last year have provided such insights into biomedically important transporters, including those required for anion exchange across the red blood cell membrane, and transporters associated with neurotransmitter uptake. Aside from the primary research into conformational changes and coupling described below, we have provide a survey of recent structural model-guided studies in the literature, focusing on proteins involved in vesicular neurotransmitter transport, in collaboration with the Schuldiner lab in Israel (ref. 1) and renal phosphate transport, in collaboration with Dr. Fenollar-Ferrer at NIMH/NIDCD (ref. 2). Studies of secondary active transporter proteins have shown that repeated elements in the structural folds of these proteins exchange between two distinct, and asymmetric, conformations. This process facilitates transport by alternately exposing binding sites to either side of the membrane, for at least 6 of the 18 known structural folds. However, it was not clear whether each and every transporter family would utilize a different, unique conformational mechanism, such as the remarkable elevator-like movement reported for the glutamate/aspartate transporters and the dicarboxylate transporter VcINDY. This year we predicted an elevator-like conformational mechanism in another transporter family, represented by the anion exchanger AE1 (also known as Band 3 or SLC4A1), which is required for exchanging chloride for bicarbonate across the membrane of red blood cells as well as the alpha-intercalated cells of the collecting ducts of the nephron. Using a method developed previously in the lab, called repeat-swap modeling, we utilized the single known structure of AE1, in which the binding site is exposed to the extracellular solution, and used it to construct a model in which the conformations of the repeats were exchanged. This model exhibited all the expected features of an inward-facing conformation, and strongly suggested that AE3 also utilizes an elevator-like conformational change (ref. 3). This work was carried out in collaboration with the Faraldo-Gomez lab (NHLBI) and Prof Michael Jennings at U. Alabama. A key feature of secondary active transporters is their ability to couple the uphill movement of one substrate to the downhill translocation of another, and thereby utilize a pre-existing concentration gradient. In amino-acid transporters of the neurotransmitter-sodium symporter (NSS) family, for example, the amino acid movement is coupled to sodium gradients. Coupling is essential, because without it the ions would dissipate their gradient, or the substrate would never capture the required driving force. In its essence, coupling means that the major outward to inward conformational change cannot occur without both substrates being bound to the protein. How does the transporter respond to this condition, while also preventing a response to each of the individual components? We have attempted to address this question for members of the NSS family: the prototypical archaeal transporter LeuT and the potassium-dependent transporter from the insect Manducta secta, KAAT1. In the case of LeuT, X-ray crystallographic structures have been determined of the outward-facing conformation in which both sodium and alanine are bound. However, it is not clear which of the many interactions formed between the substrates and the protein are required for the closing of the pathway and the conformational change of the protein toward the inward-facing conformation. We carried out molecular dynamics simulations of this structure to assess which of the interactions reported in the structure are also present under thermal fluctuations and in the presence of a hydrated lipid bilayer (as opposed to the detergent-solubilized, crystallographic form) (ref. 4). After identifying specific interactions, our collaborators in the Rudnick lab (Yale) then carried out site-specific mutagenesis of the residues involved, and assessed their effect on binding and conformational change using biochemical approaches. Together, these data revealed that one specific residue, Tyrosine-108 is responsible for translating the detection of substrate binding into the major conformational change involved in transport. Other residues that we identified, by contrast, are required exclusively for increasing the binding affinity of LeuT to one or other substrate. In the case of KAAT1, it is not clear why coupling can occur with a different driving ion, i.e. potassium instead of sodium, even though the transporter is very similar in primary sequence to LeuT. However, no structural data is available for KAAT1, and therefore we instead constructed a structural model of KAAT1 using LeuT as a template, which could then be used to predict key interactions in and around the binding sites for the two substrates (ref. 5). For the few binding site residues in this model that are unique to KAAT1, our collaborators carried out site-specific mutagenesis and electrophysiological measurements, which allowed them to identify a specific position, threonine 67, which is critical for coupling in KAAT1. In summary, our publications this year reflect ongoing efforts to utilize both modeling and computation in close collaboration with experimental laboratories, and drive understanding of the mechanism of secondary active transport related to neurotransmitter transmission and to many other biomedically important mechanisms.